Double-stranded oligonucleotide for inhibiting LPA gene expression and application of double-stranded oligonucleotide

Abstract

The invention provides double-stranded oligonucleotide for inhibiting LPA gene expression and application of the double-stranded oligonucleotide, and relates to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a positive-sense strand and an antisense strand, wherein the positive-sense strand comprises a nucleotide sequence which is the same as or basically the same as at least 15 continuous nucleotides in the sequence as shown in SEQ ID NO.1, and the antisense strand comprises at least 17 nucleotides and is complementary or basically complementary with the positive-sense strand to form a duplex region; each nucleotide of the double-stranded oligonucleotide is independently selected from an unmodified or modified nucleotide. The double-stranded oligonucleotide can inhibit LPA gene expression and can treat siRNA drugs of diseases related to LPA gene expression, such as cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present disclosure relates to the technical field of nucleic acid drugs, and in particular to a double-stranded oligonucleotide for inhibiting LPA gene expression and an application thereof. Background Art

[0002] Lipoprotein (a) (Lp(a), LPA) is a low-density lipoprotein variant. It is composed of a high molecular weight glycoprotein Apo(a) subtype homologous to plasminogen (PLG) and LDL-like particles covalently bound by disulfide bonds. It is a low-density lipoprotein-like protein with an apo(a) group. LPA is the name of the gene encoding apolipoprotein (a) (apo(a)), which is mainly expressed in the liver, and its expression is limited to humans and non-primates. Plasma Lp(a) concentration is largely determined by the LPA gene, which plays a key role in revealing the causal relationship between Lp(a) and cardiovascular disease. Lipoprotein (a) levels are the direct pathogenic factor of many cardiovascular diseases and are closely related to cardiovascular diseases such as myocardial infarction, lower limb artery disease, peripheral arteriosclerosis, and aortic valve stenosis. Mendelian randomization studies have confirmed that elevated Lp(a) levels are one of the causes of cardiovascular disease, and that Lp(a) is continuously correlated with the risk of cardiovascular disease. Among 2,100 candidate genes for cardiovascular disease, LPA gene mutations were found to be the strongest cardiovascular genetic risk factor. Therefore, the etiological role of Lp(a) in cardiovascular disease has become a research hotspot.

[0003] Based on the RNA interference mechanism, small interfering RNA (siRNA) can inhibit or block the expression of target genes in a sequence-specific manner, thereby achieving the purpose of treating diseases. Therefore, by inhibiting the expression of the LPA gene, diseases caused by abnormal lipoprotein (a) levels, especially cardiovascular diseases, can be prevented and treated at the cellular level. The guidelines for the prevention and treatment of dyslipidemia in adults define >30mg / d1 as Lp(a) abnormalities. Based on this standard, about 30% of patients with previous cardiovascular events have Lp(a) abnormalities. Although elevated Lp(a) levels are common, there is a lack of targeted therapeutic drugs. Therefore, the development of siRNA drugs that inhibit LPA gene expression and can treat diseases related to LPA gene expression will help reduce adverse cardiovascular events.

[0004] In view of this, the present disclosure is proposed. Summary of the invention

[0005] In response to the needs of cardiovascular and cerebrovascular diseases, the present disclosure uses LPA gene as a target to develop a small nucleic acid molecule drug that can reduce cholesterol levels and thus prevent or control metabolic syndrome. The first purpose of the present disclosure is to provide a double-stranded oligonucleotide and its conjugate, pharmaceutical composition, kit for inhibiting LPA gene expression, and methods and uses thereof for inhibiting or reducing LPA gene expression or treating LPA gene-mediated diseases or symptoms.

[0006] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0007] In an optional embodiment, a double-stranded oligonucleotide for inhibiting LPA gene expression is provided, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand;

[0008] Wherein, the positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO.1, wherein the substantially identical means that the nucleotide sequence has no more than 3 nucleotide differences from the at least 15 consecutive nucleotides;

[0009] Wherein, the antisense strand comprises at least 17 nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantially complementary strand means that the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides in the duplex region;

[0010] Each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0011] In an optional embodiment, a double-stranded oligonucleotide for inhibiting LPA gene expression is provided, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand;

[0012] In the direction from the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17, or at least 18 consecutive nucleotides in any one of SEQ ID NOs. 147 to 291, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides;

[0013] The sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; the substantially complementary sequence means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

[0014] In an optional embodiment, a conjugate is provided, which comprises the double-stranded oligonucleotide described above and one or more ligands capable of binding to a cell receptor.

[0015] In an optional embodiment, there is also provided the use of the above double-stranded oligonucleotide or the above conjugate in the preparation of a drug for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene.

[0016] In an optional embodiment, a pharmaceutical composition is also provided, which comprises the above-mentioned double-stranded oligonucleotide and / or the above-mentioned conjugate.

[0017] In an optional embodiment, a kit is also provided, the reagent comprising the above-mentioned double-stranded oligonucleotide, and / or the above-mentioned conjugate, and / or the above-mentioned pharmaceutical composition.

[0018] In an optional embodiment, a method for inhibiting LPA gene expression is also provided, the method comprising administering the above-mentioned double-stranded oligonucleotide, and / or the above-mentioned conjugate, and / or the above-mentioned pharmaceutical composition to a subject.

[0019] In an optional embodiment, a method for alleviating, treating and / or preventing LPA-mediated diseases or conditions is also provided, the method comprising administering the above-mentioned double-stranded oligonucleotide, and / or the above-mentioned conjugate, and / or the above-mentioned pharmaceutical composition to a subject.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The double-stranded oligonucleotides and pharmaceutical compositions provided by the present disclosure show excellent LPA gene expression regulation activity in in vitro cell experiments and in-mouse model experiments. For example, the double-stranded oligonucleotides disclosed in the present disclosure show excellent LPA mRNA inhibitory activity in LPA transgenic cell lines, and at a siRNA concentration of 1nM, the residual activity of the target gene LPA can be as low as 11.90% (RX001010). The conjugates after siRNA conjugation with ligands can have a residual activity of the target gene LPA as low as 10.05% (RZ001005, dosage 3mg / kg) and 18.76% (RZ001031, dosage 1mg / kg) in the BALB / c mouse hydrodynamic injection model. This shows that the double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates provided by the present disclosure can effectively regulate the expression level of the target gene LPA, and therefore can effectively treat and / or prevent disease symptoms related to the mRNA level expressed by the LPA gene, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 is the inhibitory activity of the target gene in BALB / c-HDI mice after administration of the siRNA conjugate in Example 4 of the present disclosure;

[0024] Figure 2 Typical histopathological images of each test group under HE staining 200 times field of view in Example 5 of the present disclosure;

[0025] Figure 3 The inhibitory activity of the target gene in BALB / c-HDI mice after administration of the siRNA conjugate in Example 6 of the present disclosure;

[0026] Figure 4 The Apo(a) protein level changes in transgenic mice after administration of siRNA conjugates in Example 8 of the present disclosure. DETAILED DESCRIPTION

[0027] The technical solution of the present disclosure will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0028] [definition]

[0029] In this document, when a letter refers to an object or quantity, uppercase and lowercase letters are distinguished, for example, Y and y represent different objects or quantities.

[0030] In this article, a "nucleotide difference" between a nucleotide sequence and another nucleic acid sequence means that the base type of the nucleotide at the same position of the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleic acid difference at this position between the two nucleic acid sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleic acid difference occurs at this position.

[0031] In this article, unless otherwise specified, capital letters A (adenine), U (uracil), G (guanine), C (cytosine), and T (thymine) represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0032] Herein, the structural formula of nucleotide is Wherein, Base represents a nucleoside base, and the nucleoside base on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0033] Herein, "2'-halogenated modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by a halogen atom. For example, a 2'-fluoro-modified nucleotide.

[0034] Herein, "2'-O-optionally substituted C1-C6 alkyl modified nucleotide" refers to a nucleotide formed by replacing the hydrogen atom on the 2' hydroxyl group of the ribose group of the nucleotide with an optionally substituted C1-C6 alkyl. Exemplarily, a 2'-O-methyl (also known as 2'-methoxy) modified nucleotide.

[0035] Herein, "2'-deoxy modified nucleotide" refers to a nucleotide in which the 2' hydroxyl group of the ribose group of the nucleotide is replaced by a hydrogen atom.

[0036] In this article, "2'-O-(CH2) n -R1 modified nucleotides refer to nucleotides in which the hydrogen atom on the 2' hydroxyl group of the ribose group is replaced by -(CH2) n -R1 replaces the formed nucleotide; illustratively, when n is selected from 1, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-O-CH2-CH3 (2'-O-ethoxymethyl) modified nucleotides or 2'-O-CH2-O-CH2-CF3 (also known as 2'-O-(2,2,2-trifluoroethoxymethyl)) modified nucleotides; when n is selected from 2, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-CH2-O-CH3 (also known as 2'-O-methoxyethyl, 2'-O-MOE) modified nucleotides. Exemplarily, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0037] Herein, "2'-O-Si(R2)3 modified nucleotide" refers to a nucleotide formed by replacing the hydrogen atom on the 2' hydroxyl group of the ribose group of the nucleotide with -Si(R4)3. Exemplary, 2'-O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide or 2'-O-TOM modified nucleotide; wherein the structural formula of TBDMS is The structural formula of TIPS is The structural formula of TOM is

[0038] Herein, a double-stranded oligonucleotide consists of two strands, of which the strand that binds to the target sequence is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand.

[0039] As used herein, the term "antisense strand" refers to that strand of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to a target sequence.

[0040] As used herein, the term "sense strand" refers to that strand of a double-stranded oligonucleotide which includes a region that is substantially complementary to a region as that term is defined herein as the antisense strand.

[0041] As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to a target sequence. Where the complementary region is not completely complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.

[0042] As used herein, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are each paired with the bases on the other strand in a complementary manner.

[0043] Herein, the terms "apolipoprotein (a) gene", Apo (a) gene, and LPA are used interchangeably in the present disclosure. LPA includes but is not limited to human LPA, cynomolgus monkey LPA, mouse LPA, and rat LPA, and its amino acid and complete coding sequence, and mRNA sequence are easily obtained using publicly available databases; its amino acid and complete coding sequence, and mRNA sequence may be naturally occurring, synthetic, recombinant, or any combination thereof.

[0044] Herein, "target sequence" refers to a continuous portion of the nucleic acid sequence of an mRNA molecule formed during transcription of LPA, including mRNA that is an RNA processing product of the primary transcription product. The targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of LPA.

[0045] Herein, the term "inhibit" can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or control (e.g., a buffer only control or an inert agent control) treated subject, cell, or sample.

[0046] As used herein, the term "small interfering RNA (siRNA)" refers to a type of double-stranded RNA comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts in the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and proteins.

[0047] As used herein, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence.

[0048] As used herein, the term "sense strand" refers to the strand of the iRNA that is substantially complementary to the antisense strand.

[0049] Herein, the term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist in the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5' end and / or 3' end of the iRNA.

[0050] Unless otherwise specified, the "compound", "ligand", "nucleic acid ligand conjugate", "siRNA conjugate", "nucleic acid", "conjugate", "chemical modification", "targeting ligand", "dsRNA", "RNAi" of the present disclosure may independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base). When it exists in the form of salt or mixed salt, it may be a pharmaceutically acceptable salt.

[0051] Herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0052] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic acid or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromates, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, analytes, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbates, salicylates, 4-aminosalicylates, and disulphonates. These salts can be prepared by methods known in the art.

[0053] As used herein, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that maintains the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, sylvite salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines, including naturally substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. These salts can be prepared by methods known in the art.

[0054] As used herein, the term "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target gene, target mRNA, or target protein-related conditions of the present invention or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a patient's target gene, target mRNA, or target protein-related condition of the present invention.

[0055] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0056] As used herein, the terms "treat," "alleviate," or "ameliorate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0057] As used herein, the terms "prevention" and "prevention" are used interchangeably to refer to an approach to obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0058] The double-stranded oligonucleotides provided in the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. The modified nucleotide groups can be introduced into the double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications. The method of preparing nucleoside monomers with corresponding modifications and the method of introducing the modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0059] As used herein, the term "chemical modification" or "modification" includes all changes in nucleic acids by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0060] As used herein, the term "base" encompasses any known DNA and RNA base, base analogs such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, hypoxanthine, and natural analogs.

[0061] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0062] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. As used herein, when the term "comprising" or "including" is used, unless otherwise indicated, the situation consisting of the elements, integers or steps described is also covered. For example, when referring to "comprising" a specific sequence, it is also intended to cover the situation consisting of the specific sequence.

[0063] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2.3-dimethylpentyl, 2.4-dimethylpentyl, 2.2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4.4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylhexyl, n-decyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and various branched chain isomers thereof, etc. In some embodiments, the alkyl group selected from the group consisting of alkyl groups having 1 to 6 carbon atoms includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. By way of example only, "C1-4 alkyl" means having one to four carbon atoms in the alkyl chain, i.e., the C1-4 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. The alkyl group may be substituted or unsubstituted.

[0064] As used herein, the term "alkoxy" refers to -O-alkyl, wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. By way of example only, "C 1-10 "Alkylene" means an alkyl group having one to ten carbon atoms in the alkyl chain. 1-10The alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), and the like.

[0065] As used herein, the term "hydroxyl" refers to an -OH group.

[0066] Herein, the term "halogen" or "halo" refers to any of the radioactive-stable atoms in column 7 of the periodic table, such as fluorine, chlorine, bromine or iodine, among which fluorine and chlorine are preferred, and fluorine is more preferred.

[0067] As used herein, the term "connection", when referring to a connection between two molecules, refers to two molecules being connected by a covalent bond or two molecules being associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct connection and indirect connection. The term "direct connection" refers to a first compound or group being connected to a second compound or group without any intervening atoms or atomic groups. The term "indirect connection" refers to a first compound or group being connected to a second compound or group via an intermediate group, compound or molecule (e.g., a linking group).

[0068] As used herein, the term "optionally substituted" is used to define a variable that can be unsubstituted or substituted.

[0069] As used herein, the term "unsubstituted" means that the designated group bears no substituents.

[0070] As used herein, the terms "substituted," "substituted," and "substituted" are used interchangeably to indicate that any one or more hydrogen atoms in a given structure are replaced by a specified substituent (e.g., C 1-3 Alkyl, C 1-3 alkoxy or halogen), provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structural formula can be substituted by one or more substituents selected from a specific group, then the substituents may be substituted the same or differently at each substitutable position.

[0071] Herein, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0072] In the structural formulas of "compounds", "ligands" and "supports", the bond "-" indicates an unspecified configuration. If chiral isomerism exists in the chemical structure, the bond "-" can be or include both Although all of the above structural formulae are drawn in certain isomeric forms for simplicity, the present disclosure may include all isomers, for example, tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0073] As used herein, the term "stereoisomers" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0074] As used herein, the term "chiral" refers to a molecule that has the property of being non-superimposable on its mirror image; whereas "achiral" refers to a molecule that is superimposable on its mirror image.

[0075] As used herein, the term "enantiomers" refers to two isomers of a compound that are non-superimposable but mirror images of each other.

[0076] As used herein, the term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.

[0077] As used herein, stereochemical definitions and conventions generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0078] Herein, the term "conjugation" refers to the covalent connection between two or more chemical moieties each having a specific function; accordingly, "conjugate" refers to a compound formed by covalent connection between two or more chemical moieties. Further, "drug conjugate" refers to a compound formed by covalently connecting one or more chemical moieties with specific specific functions to an active drug. Herein, sometimes, especially in the embodiments, the "drug conjugate" of the present disclosure is also referred to as a "conjugate", "double-stranded oligonucleotide conjugate" or "siRNA conjugate". Drug conjugate should be understood as a general term for drug conjugates or a specific drug conjugate represented by a specific structural formula, depending on the context.

[0079] In the first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression, wherein the double-stranded oligonucleotide can inhibit LPA gene expression. The double-stranded oligonucleotide can inhibit LPA gene expression in cells in vitro, and can also inhibit LPA gene expression in the body in vivo.

[0080] The double-stranded oligonucleotide includes a sense strand and an antisense strand.

[0081] Wherein, the positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO.1, and the substantially identical means that the nucleotide sequence has no more than 3 (e.g., 0, 1, 2 or 3) nucleotide differences with the at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) consecutive nucleotides.

[0082] In an optional embodiment, the positive strand comprises 15, 16, 17, 18, 19 or 20 consecutive nucleotides in the SEQ ID NO.1 sequence or a nucleotide sequence having no more than 2 (eg, 0, 1 or 2) nucleotide differences with the consecutive nucleotides.

[0083] Wherein, the antisense strand comprises at least 17 (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28) nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantially complementary strand means that the sense strand and the antisense strand have no more than 3 (e.g., 0, 1, 2 or 3) nucleotide mismatches in the duplex region.

[0084] In an optional embodiment, the antisense strand is complementary or substantially complementary to 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the nucleotide sequence of SEQ ID NO.1, and the substantially complementary means that the mismatch in the complementary region does not exceed 3 (e.g., 0, 1, 2 or 3) nucleotides.

[0085] In alternative embodiments, the length of the duplex region of the double-stranded oligonucleotide is 15-25, 15-23, 15-21, 15, 19, 15-17, 16-25, 16-24, 16-22, 16-20, 16-18, 17-25, 17-23, 17-22, 17-20, 17-19, 18-25, 18-24, 18-23, 18-21, 18-20, 19-25, 19-23, 19-21, 20-25, 20-24, 20-22, 21-25, 21-23, 22-25, 22-24 or 23-25 ​​nucleotide pairs. The length of the duplex region of the double-stranded oligonucleotide is more specifically, for example but not limited to, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs.

[0086] In an alternative embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0087] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Wherein, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most but not all of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0088] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotide, wherein n is selected from 1 or 2, and R1 is selected from an optionally substituted C1-C6 alkyl or an optionally substituted C1-C6 alkoxy. If R1 contains a substituent, the substituent in R1 means that one or more hydrogen atoms on the C1-C6 alkoxy or C1-C6 alkoxy are replaced by a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0089] In an optional embodiment, the 2'-O-(CH2) nThe -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0090] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0091] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0092] In a second aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand;

[0093] From the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17 or at least 18 consecutive nucleotides in any one of SEQ ID NOs. 147 to 291, or a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides.

[0094] In the direction from the 5' end to the 3' end, the sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 1, 2 or 3 nucleotides.

[0095] In an optional embodiment, the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of any sequence in SEQ ID NO.147-291, or a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides; in an optional embodiment, the antisense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences in any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences in any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand comprises a nucleotide sequence having 0 or 1 nucleotide difference in any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand is selected from or comprises any one of the nucleotide sequences in SEQ ID NO.147-291.

[0096] In an optional embodiment, the nucleotide sequence of the sense chain comprises at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides of any one of SEQ ID NO.2 to 146, or has 1, 2 or 3 nucleotide differences with the consecutive nucleotides; in an optional embodiment, the nucleotide sequence of the sense chain has a nucleotide sequence with 0, 1, 2 or 3 nucleotide differences with any one of SEQ ID NO.2 to 146; in an optional embodiment, the nucleotide sequence of the sense chain comprises a nucleotide sequence with 0, 1 or 2 nucleotide differences with any one of SEQ ID NO.2 to 146; in an optional embodiment, the nucleotide sequence of the sense chain comprises a nucleotide sequence with 0 or 1 nucleotide difference with any one of SEQ ID NO.2 to 146; in an optional embodiment, the nucleotide sequence of the sense chain is selected from or comprises any one of SEQ ID NO.2 to 146.

[0097] In an optional embodiment, the double-stranded oligonucleotide is selected from a modified or unmodified double-stranded oligonucleotide shown in any number from RN001001 to RN001145 in Table 1.

[0098] In an optional embodiment, the double-stranded oligonucleotide is selected from one or more of the modified or unmodified double-stranded oligonucleotides shown in the following numbers: RN001008, RN001010, RN001011, RN001025, RN001042, RN001074 and RN001121.

[0099] Table 1

[0100]

[0101]

[0102]

[0103] In an alternative embodiment, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0104] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Wherein, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most but not all of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0105] In an alternative embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides.

[0106] In an optional embodiment, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides:

[0107] 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-optionally substituted C1-C6 alkyl modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA) and unlocked nucleic acid (UNA).

[0108] If the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide contains a substituent, the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide means that one or more hydrogen atoms in the C1-C6 alkyl are replaced by a substituent, and the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0109] In an optional embodiment, the 2'-halogenated modified nucleotides are, for example but not limited to, selected from 2'-fluoro-modified nucleotides.

[0110] In an optional embodiment, the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is, for example but not limited to, selected from 2'-O-methyl modified nucleotides.

[0111] In an optional embodiment, the 2'-O-(CH2) n -O-R1 modified nucleotides, n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy;

[0112] If R1 contains substituents, the substituents in R1 refer to C1-C6 alkyl or one or more hydrogen atoms in the C1-C6 alkyl are replaced by substituents, and the substituents in R1 are independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0113] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0114] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0115] In an optional embodiment, each R2 in the 2'-O-Si(R2)3 modified nucleotide is independently selected from an unsubstituted or substituted C1-C6 alkyl group;

[0116] If R2 contains a substituent, the substituent in R2 refers to one or more hydrogen atoms in the C1-C6 alkyl group being replaced by a substituent, and the substituent in R2 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0117] In an optional embodiment, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide.

[0118] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotide or 2'-O-Si(R2)3 modified nucleotide.

[0119] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0120] In an optional embodiment, the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0121] In an optional embodiment, the sense strand contains at most two 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0122] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides or 2'-O-methoxyethyl modified nucleotides.

[0123] In an alternative embodiment, the sense strand or the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0124] In an alternative embodiment, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0125] In an alternative embodiment, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0126] In an alternative embodiment, the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0127] In an optional embodiment, the sense strand is 18, 19, 20 or 21 nt in length, and the antisense strand is 19, 20, 21, 22 or 23 nt in length.

[0128] In an optional embodiment, the sense strand is 19 nt in length, and the antisense strand is 21 nt in length.

[0129] In an alternative embodiment, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds.

[0130] In an alternative embodiment, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0131] In an alternative embodiment, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

[0132] In an optional embodiment, the structure of the duplex region of the double-stranded oligonucleotide is as shown in formula (101):

[0133] SS: 5'-(N)a'-(X)i'-(N)b'-(X)t'-(N)c'-(X)r'-(N)d'-3'

[0134] AS: 3'-(N)a-(X)i-(N)b-(X)t-(N)c-5' formula (101);

[0135] Among them, SS represents the sense strand and AS represents the antisense strand;

[0136] Each N is independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, or a 2'-deoxy modified nucleotide;

[0137] Each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotide or 2'-O-Si(R2)3 modified nucleotide; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; each R2 is independently selected from optionally substituted C1-C6 alkyl; in an optional embodiment, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides. In an optional embodiment, each X is independently selected from 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides.

[0138] a, a', i, i', b, b', t, t', c, c', r', d' independently represent the number of nucleotides; wherein, a' is selected from an integer of 3-8, i' is selected from an integer of 0-3, b' is selected from an integer of 4-13, t' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, i is selected from an integer of 0-1, b is selected from an integer of 4-8, t is selected from an integer of 0-4, and c is selected from an integer of 6-10; and, i', t', r', i, t are not 0 at the same time, and 0≤t'+r'≤4. In an optional embodiment, a' is selected from an integer of 3-8, i' is selected from 0 or 1, b' is selected from an integer of 4-13, t' is selected from 0 or 1, and c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, i is selected from 1, b is selected from an integer of 4-8, t is selected from 0 or 1, and c is selected from an integer of 6-10.

[0139] In an alternative embodiment, each N is independently selected from a 2'-fluoro modified nucleotide or a 2'-O-methyl modified nucleotide.

[0140] In an alternative embodiment, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0141] In an alternative embodiment, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide.

[0142] In an alternative embodiment, the duplex region represented by formula (101) comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide.

[0143] In an optional embodiment, the sense strand length of the double-stranded oligonucleotide is 18-21, 18-20, 18-19, 19-21, 19-20 or 20-21 nucleotides; more specifically, the sense strand length of the double-stranded oligonucleotide having the duplex region shown in formula (101) is 17, 18, 19, 20 or 21 nucleotides.

[0144] In an optional embodiment, the antisense strand length of the double-stranded oligonucleotide is 19-23, 19-22, 19-21, 19-20, 20-23, 20-22, 20-21, 21-23, 21-22 or 22-23 nucleotides; more specifically, the antisense strand length of the double-stranded oligonucleotide is 19, 20, 21, 22 or 23 nucleotides.

[0145] In an optional embodiment, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

[0146] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0147] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, at most two nucleotides among the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11, and 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0148] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, at most two nucleotides among the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0149] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0150] In an optional embodiment, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0151] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide, wherein the modification of the sense strand and the antisense strand is selected from any one of the following (1)-(10):

[0152] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0153] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0154] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0155] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0156] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0157] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0158] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0159] (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 9 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide.

[0160] (9) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide.

[0161] (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

[0162] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from:

[0163] In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0164] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from:

[0165] In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0166] In an optional embodiment, in the direction from the 5' end to the 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds.

[0167] In an optional embodiment, the 5' end and the 3' end of the antisense strand independently contain 1 or 2 consecutive phosphorothioate bonds.

[0168] In an optional embodiment, in the direction from the 5' end to the 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds, and the 5' end and 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

[0169] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and the sense strand is selected from any one of the sense strands A1 to A11 listed in Table 2; and / or, the antisense strand is selected from any one of the antisense strands B1 to B41 listed in Table 2.

[0170] In an optional embodiment, the double-stranded oligonucleotide is selected from any one of RX001149 to RX001192 in Table 2:

[0171] Table 2

[0172]

[0173]

[0174]

[0175] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0176] In the third aspect of the present disclosure, the present disclosure further provides a conjugate, which comprises the double-stranded oligonucleotide described in the first aspect or the double-stranded oligonucleotide described in the second aspect, and one or more ligands capable of binding to cell surface receptors.

[0177] In an optional embodiment, the 3' end and / or the 5' end of the sense strand and / or the antisense strand is conjugated to the ligand.

[0178] In an alternative embodiment, the 3' end and / or the 5' end of the sense strand is conjugated to the ligand.

[0179] In an alternative embodiment, the 3' end of the sense strand is conjugated to the ligand.

[0180] In an optional embodiment, the ligand is linked to the 3' end and / or 5' end of the sense strand and / or antisense strand via a phosphate group or a phosphorothioate group.

[0181] In an optional embodiment, the number of the ligands includes but is not limited to 1, 2, 3 or 4.

[0182] In an optional embodiment, the number of the ligands is selected from 1.

[0183] In an optional embodiment, the number of the ligands is selected from 1, and one of the ligands is conjugated to the 3' end of the sense strand.

[0184] In an alternative embodiment, the ligand is specifically targeted to liver tissue.

[0185] In an optional embodiment, the ligand is selected from asialoglycoprotein receptor ligands.

[0186] In an alternative embodiment, the ligand comprises galactose or a derivative thereof.

[0187] In an optional embodiment, the galactose derivative is selected from N-acetylgalactosamine.

[0188] In an optional embodiment, the structure of the conjugate is shown in formula (201):

[0189]

[0190] Wherein, Nu represents the double oligonucleotide, each R ligand Independently selected from ASGPR ligands, y is selected from 1, 2, 3 or 4.

[0191] Each of the R ligand are independently conjugated to the 3' end and / or 5' end of the sense strand and / or the antisense strand, and each of the R ligand The conjugation positions are different.

[0192] In an optional embodiment, y is selected from 1 or 2.

[0193] In an optional embodiment, y is selected from 1.

[0194] In an optional embodiment, y is selected from 1, and one of said R ligand Conjugated to the 3' end of the sense strand.

[0195] In an optional embodiment, y is selected from 2.

[0196] In an optional embodiment, y is selected from 2, and the two R ligand conjugated to the 3' and 5' ends of the sense strand, respectively.

[0197] In an optional embodiment, each R ligand Each is independently selected from the structure represented by formula (301) or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0198]

[0199] Wherein, * represents the conjugation site of the ligand on the sense strand or the antisense strand.

[0200] In formula (301), j is selected from 1, 2, 3 or 4, further optionally, j is selected from 1, 2 or 3; further optionally, j is selected from 3.

[0201] In formula (301), each Z is independently selected from hydroxyl or thiol; further optionally, Z is selected from hydroxyl.

[0202] In formula (301), each p is independently selected from 1, 2 or 3; further optionally, p is selected from 1.

[0203] In formula (301), each q is independently selected from 1, 2 or 3; further optionally, q is selected from 1.

[0204] In formula (301), each R3 is independently selected from H, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy. If R3 contains a substituent, the substituent in R3 means that one or more hydrogen atoms in the C1-C6 alkyl or C1-C6 alkoxy are replaced by a substituent, and the substituent in R3 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0205] Further optionally, in formula (301), each R3 is selected from H.

[0206] In formula (301), each L is independently selected from an optionally substituted C1-C 20 Alkylene or R La and R La are independently selected from optionally substituted C1-C 10 Alkylene, k is selected from 1, 2, 3, 4 or 5. If L contains a substituent, the substituent in L refers to C1-C 20 Alkane or C1-C 10 One or more hydrogen atoms in the alkylene group are substituted by a substituent, and the substituent in L is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0207] In an optional embodiment, each L is independently selected from C1-C 10 Alkylene or Among them, R La and R La are independently selected from C1-C5 alkylene, and k is 1, 2 or 3. Further optionally, k is selected from 1.

[0208] In an optional embodiment, each L is independently selected from

[0209] In an optional embodiment, each L is independently selected from

[0210] In an optional embodiment, L is selected from

[0211] In an optional embodiment, L is selected from

[0212] In an alternative embodiment, each Y is independently selected from O, S or NH.

[0213] In an optional embodiment, Y is selected from O.

[0214] In an optional embodiment, each Rligand Independently selected from the structure shown in formula (302) or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0215]

[0216] In an optional embodiment, each R ligand Independently selected from any of the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0217]

[0218]

[0219] In an optional embodiment, each R ligand Independently selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0220]

[0221] In an optional embodiment, the structural formula of the conjugate is as shown below or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0222]

[0223] in, represents the double-stranded oligonucleotide (eg siRNA). Further, the ligand (CR01008×3) is conjugated to the 3′ end of the sense strand of the double-stranded oligonucleotide via a phosphodiester bond.

[0224] In some other optional embodiments, each R ligand Independently selected from the following structures or pharmaceutically acceptable salts thereof:

[0225]

[0226] In an optional embodiment, Nu in formula (201) is selected from any one of RX001149 to RX001192 in Table 2.

[0227] In an optional embodiment, Nu is selected from any one of RX001149 to RX001192 in Table 2, the ligand is selected from the formula (CR01008×3), y=1, and one of the ligands is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0228] In an optional embodiment, the conjugate is selected from the double-stranded oligonucleotide conjugates shown by any number from RZ001031 to RZ001074 in Table 3.

[0229] Table 3

[0230]

[0231]

[0232]

[0233] In the above table, it is explained exemplarily that the positive strand GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm_(CR01008×3) in RZ001031 indicates that the ligand (CR01008×3) is conjugated to the 3' end of the sequence GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm_(CR01008×3) as shown in SEQ ID NO.307.

[0234] In the fourth aspect of the present disclosure, the present disclosure provides the use of the double-stranded oligonucleotide described in the first aspect, and / or the double-stranded oligonucleotide described in the second aspect, and / or the conjugate described in the third aspect in the preparation of a drug for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene.

[0235] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect.

[0236] In the sixth aspect of the present disclosure, the present disclosure provides a kit, which comprises the double-stranded oligonucleotide described in the first aspect, and / or the double-stranded oligonucleotide described in the second aspect, and / or the conjugate described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0237] In the seventh aspect of the present disclosure, the present disclosure provides a method for inhibiting LPA gene expression, the method comprising administering to a subject the double-stranded oligonucleotide described in the first aspect, and / or the double-stranded oligonucleotide described in the second aspect, and / or the conjugate described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0238] In the eighth aspect of the present disclosure, the present disclosure provides a method for alleviating, treating and / or preventing LPA-mediated diseases or conditions, the method comprising administering to a subject the double-stranded oligonucleotide described in the first aspect, and / or the double-stranded oligonucleotide described in the second aspect, and / or the conjugate described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0239] The LPA-mediated diseases or disorders in any of the above aspects include diseases associated with LPA gene expression.

[0240] In an alternative embodiment, the LPA gene-mediated disease or condition comprises cardiovascular disease.

[0241] In an optional embodiment, the cardiovascular disease includes but is not limited to: hyperlipoprotein(a)emia, Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoproteinβ-lipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or pathology associated with elevated levels of Lp(a) particles.

[0242] In an optional embodiment, the double-stranded oligonucleotide and / or the conjugate in any of the above embodiments; or, the double-stranded oligonucleotide and / or the conjugate in the pharmaceutical composition is administered in an unbuffered solution or a buffer.

[0243] In alternative embodiments, the unbuffered solution is saline or water.

[0244] In an optional embodiment, the buffer component in the buffer comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof.

[0245] In an alternative embodiment, the buffer is phosphate buffered saline (PBS).

[0246] In an optional embodiment, the subject of the double-stranded oligonucleotide in any of the above embodiments and / or the above conjugate is a cell.

[0247] In an optional embodiment, the subject is selected from cells, comprising substantially contacting the subject cells with the double-stranded oligonucleotide, and / or the conjugate, and / or the pharmaceutical composition.

[0248] In an optional embodiment, the subject is selected from cells, and the administration method comprises: (a) contacting the cells with the above-mentioned double-stranded oligonucleotide, and / or the above-mentioned conjugate, and / or the above-mentioned pharmaceutical composition; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the LPA gene, thereby inhibiting the expression of the LPA gene in the cells.

[0249] In an optional embodiment, the subject is selected from humans.

[0250] In an alternative embodiment, the subject is selected from cells derived from humans.

[0251] In an optional embodiment, the inhibition of LPA gene expression is the inhibition of LPA gene expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%. The inhibition rate of LPA gene expression can be determined by conventional methods known in the art, such as but not limited to luciferase reporter gene assay, PCR method or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0252] In an optional embodiment, the double-stranded oligonucleotide and / or the conjugate is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

[0253] In an alternative embodiment, the double-stranded oligonucleotide and / or the conjugate is administered subcutaneously or intravenously.

[0254] The present disclosure is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present disclosure in any form.

[0255] Unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the synthesis of PCR primers used in the present disclosure was commissioned to Sangon Biotechnology (Shanghai) Co., Ltd.; the HEK293-LPA stable cell line used in the present disclosure was donated by Beijing Institute of Technology; the experimental animal BALB / c mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; the experimental animal ICR mice used in the present disclosure were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; the hApo(a) transgenic mice used in the present disclosure were constructed by Suzhou Xuanjing Biotechnology Co., Ltd. and commissioned by Saiye (Suzhou) Biotechnology Co., Ltd.; the transgenic mouse serum samples in the present disclosure were detected using a human Lipoprotein A ELISA kit.

[0256] Unless otherwise stated, the real-time PCR detection data of the activity experiments involved in the present disclosure all use the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0257] ΔCt(test group)=Ct(test group target gene)-Ct(test group reference gene);

[0258] ΔCt(control group)=Ct(control group target gene)-Ct(control group internal reference gene);

[0259] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average);

[0260] ΔΔCt(control group)=ΔCt(control group)-ΔCt(control group average).

[0261] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%;

[0262] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (test group) × 100%;

[0263] The target gene mRNA inhibition rate of the test group = 100% - the relative expression level of the target gene mRNA of the test group;

[0264] The detection data of Apo(a) protein at different time points in the in vivo activity experiment involved in the present disclosure and the calculation method are summarized as follows:

[0265]

[0266] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0267] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0268] Preparation of compounds

[0269] Unless otherwise specified, the reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. The information of the main reagents is shown in Table 4.

[0270] Table 4

[0271]

[0272] Among them, CPG stands for controlled pore glass (Controlled Pore Glass) carrier.

[0273] Preparation Example 1 Preparation of Compound CR01008

[0274] (1.1) The synthetic structure of compound CR01008 is shown below:

[0275]

[0276] The synthetic route of compound CR01008 is as follows:

[0277]

[0278] (1.1.1) Synthesis of compound 2

[0279] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde aqueous solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of KOH aqueous solution with a concentration of 45.3% by mass was added dropwise. After the addition was completed, the reaction was stirred at 25°C for 30 minutes, the temperature was raised to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude product as a white solid. A small amount of water was added to the crude product for pulping, and filtered to obtain compound 2 as a white solid (9 g, yield 78.9%). MS-ESI (m / z) = 260 [M+H] + .

[0280] (1.1.2) Synthesis of compound 3

[0281] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 ml of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (45 ml, 4 M) was added, and the mixture was stirred at 25°C for 1 hour. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (6.8 g, yield 100%) as a white solid.

[0282] (1.1.3) Synthesis of compound 5

[0283] Compound 3 (1.8 g, 2.0 eq) prepared in step (1.1.2), compound 4 (5-[[(2R, 3R, 4R, 5R, 6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq) and DIEA (N, N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 ml of DMF, HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25° C. in a N2 atmosphere for 3 hours. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure and reversely purified (22% by volume acetonitrile aqueous solution) to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0284] (1.1.4) Synthesis of compound 6

[0285] The compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C, reacted at 25°C for 3 hours, and 15 ml of methanol was added to the reaction solution to quench the reaction. After the reaction was completed, the reaction solution was evaporated under reduced pressure and reversely purified (60% by volume of acetonitrile in water) to obtain compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0286] (1.1.5) Synthesis of compound CR01008

[0287] The compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added respectively, and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25°C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined, evaporated to dryness under reduced pressure, reversely purified (72% by volume acetonitrile aqueous solution), and then vacuum dried for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0288] H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58- 3.39(m,3H),3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0289] (1.2) Synthesis of compound CR01008Z

[0290]

[0291] The synthetic route of compound CR01008Z is as follows:

[0292]

[0293] (1.2.1) Synthesis of compound 9

[0294] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg) and TEA (226.2 mg) were added. The nitrogen atmosphere was replaced three times, and the mixture was stirred at 25°C for 16 hours. The mixture was flash purified to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0295] (1.2.2) Synthesis of compound CR01008Z

[0296] Compound 9 (50 mg, aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample bottle and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V) and Cap2 (1 ml, 20 V) were added to a 20 ml sample bottle and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0297] Wherein, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20 volume % acetic anhydride acetonitrile solution.

[0298] Preparation Example 2 Preparation of Compound CR01013

[0299] (2.1) Synthesis of compound CR01013

[0300]

[0301] The synthetic route of compound CR01013 is as follows:

[0302]

[0303] (2.1.1) Synthesis of compound 2

[0304] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, concentration of 37 mass %) and aqueous sodium hydroxide solution (6.5 ml, concentration of 45.3 mass %) were added dropwise. After the addition was completed, the temperature was raised to 60°C and stirred at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C and evaporated to dryness under reduced pressure to obtain a crude product as a white solid. A small amount of water was added to the crude product for pulping, and the product was filtered and dried to obtain compound 2 as a white solid (4.8 g, yield 85.9%). ESI-MS (m / z) = 260.2 [M+H] + .

[0305] (2.1.2) Synthesis of compound 3

[0306] Compound 2 (4.8 g) prepared in step (2.1.1) was dissolved in 25 ml of 1,4-dioxane, and a 1,4-dioxane solution of hydrochloric acid (25 ml, 4 M) was added, and the mixture was stirred at 25° C. for 2 hours. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (3.6 g, yield 99.4%) as a white solid.

[0307] (2.1.3) Synthesis of compound 11

[0308] Compound 3 (3.6 g) prepared in step (2.1.2) was dissolved in 36 ml of DMF, and then TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added and stirred at 25°C for 16 hours. After the reaction, the reaction solution was added to 200 ml of saturated sodium bicarbonate aqueous solution, extracted three times with 100 ml of ethyl acetate (3×100 ml), and the organic phases were combined. The organic phase was first washed once with 50 ml of saturated sodium chloride aqueous solution (1×50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by column chromatography (eluent: dichloromethane / methanol=10 / 1, v / v) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESIMS (m / z)=379.5 [M+H] + .

[0309] (2.1.4) Synthesis of compound 12

[0310] The compound 11 (2.3 g) prepared in step (2.1.3) was dissolved in 23 ml of methanol, wet palladium carbon (230 mg, loading 10 mass%) was added, and hydrogen was replaced three times. The reaction system was stirred at 25° C. in a hydrogen atmosphere (15 psi) for 16 hours. After the reaction was completed, the reaction solution was filtered to obtain a filtrate, and the filtrate was evaporated under reduced pressure to obtain a yellow oily compound 12 (1.48 g, yield 99.8%).

[0311] (2.1.5) Synthesis of compound 13

[0312] The compound 12 (1.48 g) prepared in step (2.1.4) was dissolved in 15 ml of DMF, and triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added, and the mixture was stirred at 25°C for 16 hours. After the reaction, the reaction solution was added to 150 ml of saturated sodium bicarbonate aqueous solution, and extracted three times with 50 ml of ethyl acetate (3×50 ml), and the organic phases were combined. The organic phase was first washed once with 30 ml of saturated sodium chloride aqueous solution (1×30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure, and purified by column chromatography (C18 column, eluent: water / acetonitrile=5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M+H] + .

[0313] (2.1.6) Synthesis of compound 14

[0314] The compound 13 (1.1 g) prepared in step (2.1.6) was dissolved in 11 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (813 mg) was added in batches at 0°C, and the reaction system was stirred at 0°C for 1 hour. After the reaction was completed, methanol was added to the reaction solution to quench, the solvent was evaporated, and the reaction solution was purified by reverse phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z): 976.5 [M+H] + .

[0315] (2.1.7) Synthesis of compound CR01013

[0316] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added, and the nitrogen atmosphere was replaced three times. The reaction system was stirred at 25°C for 1 hour in a nitrogen atmosphere. After the reaction was completed, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate aqueous solution (2×5 ml), and then washed once with 30 ml of saturated sodium chloride aqueous solution (1×30 ml), the organic phase was separated, dried over anhydrous sodium sulfate, and the organic phase solvent was evaporated under reduced pressure. The compound was purified by column chromatography (eluent: dichloromethane / methanol=20 / 1, v / v) to obtain compound CR01013 (532 mg, yield 80.4%) as a white solid. ESI-MS (m / z): 1176.7 [M+H] + .

[0317] 1H NMR (400MHz, DMSO-d6) δ0.95-1.05 (d, J=6.7Hz, 5H), 1.06-1.15 (q, J=7.6Hz, 8H), 1.15-1.21 (t, J=7.2Hz, 14H), 1.72-1 .80(s,3H),1.84-1.92(s,3H),1.94-2.07(d,J=16.0Hz,7H),2.07-2.14(s,3H),2.64-2.72(q,J=5.8Hz,2H),2.74-2.89 (d,J=8.5Hz,2H),3.35-3.56(m,4H),3.57-3.70(m,4H),3.71-3.77(s,6H),3.81-3.93(m,1H),3.96-4.09(d,J=6.4Hz, 3H), 6.82-6.97 (d, J=8.7Hz, 4H), 7.17-7.27 (t, J=8.7Hz, 5H), 7.27-7.34 (t, J=7.6Hz, 2H), 7.34–7.43 (d, J=7.5Hz, 2H).

[0318] (2.2) Synthesis of compound CR01013Z

[0319]

[0320] The synthetic route of compound CR01013Z is as follows:

[0321]

[0322] (2.2.1) Synthesis of compound 15:

[0323] At 25°C, compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane, triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added, and the reaction system was stirred at 25°C for 16 hours. After the reaction, the solvent in the reaction solution was evaporated, and column chromatography was performed for reverse phase purification (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain yellow oily compound 15 (110 mg, 0.10 mmol, yield 100%). ESI-MS (m / z) = 1099.3 [M+Na] +.

[0324] (2.2.2) Synthesis of compound CR01013Z:

[0325] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino-CPG (1.06 g, loading 80 μmol / g) were added. The reaction system was stirred at 25°C for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed twice with 50 ml of dichloromethane (2×50 ml), three times with 50 ml of acetonitrile (3×50 ml), and once with 50 ml of ethyl acetate (1×50 ml), and then vacuum dried. Cap1 (4.8 ml), Cap2 (0.54 ml) and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system was stirred at 25°C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3×50 ml) and vacuum dried to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0326] Wherein, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20 volume % acetic anhydride acetonitrile solution.

[0327] Preparation of siRNA / siRNA conjugates

[0328] Preparation Example 3 Preparation of siRNA

[0329] (3.1) Synthesis of the positive strand (SS)

[0330] By the method of phosphoramidite nucleic acid solid phase synthesis, the compound of the solid phase carrier is connected to the nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0331] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0332] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0333] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the thiolation reagent is an acetonitrile / pyridine mixed solution of hydrogenated xanthanol with a concentration of 0.2M (the volume ratio of acetonitrile and pyridine is 1:1).

[0334] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a volume ratio of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0335] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; thiolation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0336] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0337] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0338] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0339] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the positive strand of siRNA is obtained.

[0340] (3.2) Synthesis of antisense strand (AS)

[0341] The antisense chain was synthesized using a universal solid phase carrier. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (3.1) for synthesizing the sense chain.

[0342] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the antisense strand of siRNA is obtained.

[0343] (3.3) siRNA synthesis

[0344] The sense chain synthesized in step (3.1) and the antisense chain synthesized in step (3.2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and the antisense chain shown in the aforementioned Table 2 and the following Table 5.

[0345] Table 5 siRNA sequence information

[0346]

[0347]

[0348]

[0349]

[0350]

[0351] In the above table, the modified nucleotide sequence shown in the positive chain of RX000001 is the naked sequence shown in SEQ ID NO.305, and the modified nucleotide sequence shown in the antisense chain of RX000001 is the naked sequence shown in SEQ ID NO.306.

[0352] Preparation Example 4 Preparation of siRNA conjugates in which the 3' end of the sense strand is conjugated to the L96 vector

[0353] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd., with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is shown in the following formula (R-L96-PS):

[0354]

[0355] Wherein, PS represents polystyrene resin solid phase carrier.

[0356] (4.1) Synthesis of positive chain

[0357] By the method of phosphoramidite nucleic acid solid phase synthesis, the L96-PS compound connected to the solid phase carrier is used to start the cycle, and the nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis method and conditions refer to the positive chain synthesis method shown in step (3.1) of Preparation Example 3.

[0358] (4.2) Synthesis of antisense strand

[0359] The antisense strand of the siRNA conjugate was synthesized according to the antisense strand synthesis method shown in step (3.2) of Preparation Example 3.

[0360] (4.3) Synthesis of siRNA conjugates

[0361] The siRNA conjugate in which the 3' end of the sense strand is conjugated to the L96 vector in this preparation example was synthesized according to the siRNA synthesis method shown in step (3.3) of preparation example 3. The structural formula of the siRNA conjugate in which the 3' end of the sense strand is conjugated to the L96 vector is as follows:

[0362]

[0363] in, Represents siRNA. L96 is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0364] Table 6 Sequence information of siRNA conjugates with the 3' end of the sense strand conjugated to the L96 vector.

[0365]

[0366]

[0367] In the above table, the modified nucleotide sequences shown in the sense strands of RZ000001 and RZ000002 correspond to the naked sequence shown in SEQ ID NO.301, and the modified nucleotide sequences shown in the antisense strands of RZ000001 and RZ000002 correspond to the naked sequence shown in SEQ ID NO.302.

[0368] Explained illustratively, the positive strand UmsUmsCmUmCmCmGfAfAfCmGmUmGmUmCmAmCmGmUm_L96 in number RZ000001 indicates that the ligand L96 is conjugated to the 3' end of the sequence UmsUmsCmUmCmCmGfAfAfCmGmUmGmUmCmAmCmGmUm as shown in SEQ ID NO.648 via a phosphodiester bond.

[0369] Preparation Example 5 Synthesis of siRNA conjugates with the 3' end of the sense strand conjugated to a carrier (CR01008×3)

[0370] (5.1) Synthesis of the positive strand of the 3' end conjugated (CR01008×3) vector

[0371] By the phosphoramidite nucleic acid solid phase synthesis method, starting from the compound CR01008Z connected to the solid phase carrier, the nucleoside monomers are connected one by one in a 3'-5' direction according to the nucleotide sequence. During the synthesis process, the compound CR01008 is regarded as a nucleoside monomer.

[0372] Each connection of a compound includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the synthesis of the sense chain of this preparation example are the same as those in the synthesis of the sense chain in step (3.1) of preparation example 3.

[0373] In the synthesis process of the positive chain of the present disclosure, three clusters of CR01008 are obtained, which are recorded as (CR01008×3) or (CR01008)×3.

[0374] The structural formula of the three-cluster CR01008 is as follows:

[0375]

[0376] (5.2) Synthesis of antisense strand

[0377] The antisense strand of this preparation example was synthesized according to the antisense strand synthesis method shown in step (3.2) of preparation example 3.

[0378] (5.3) siRNA conjugates with the 3' end of the sense strand conjugated to the (CR01008×3) carrier The siRNA conjugates with the 3' end of the sense strand conjugated to the (CR01008×3) carrier in Table 3 were synthesized according to the method described in step (3.3) of Preparation Example 3.

[0379] The structural formula of the siRNA conjugate of the 3' end of the positive chain conjugated to the (CR01008×3) carrier is as follows:

[0380]

[0381] in, Represents siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0382] In the sequence disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a 2'-deoxy-modified nucleotide (also known as deoxynucleotide); lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond; the combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide.

[0383] Biological detection experiments

[0384] Unless otherwise specified, the reagents, consumables and instruments used in the biological detection experiments of the present disclosure are all from commercially available products. Among them, the main reagents and consumables and their sources are shown in Table 7, and the main instruments and their sources are shown in Table 8.

[0385] Table 7 Main reagents and consumables

[0386] name factory 1× PBS Zhongke Maichen (Beijing) Technology Co., Ltd. DMEM medium Zhongke Maichen (Beijing) Technology Co., Ltd. <![CDATA[Opti-MEM TM Culture medium]]> Gibco Fetal bovine serum Sigma Pancreatin Zhongke Maichen (Beijing) Technology Co., Ltd. Dual Antibody BBI Lipofectamine RNAiMax Invitrogen Nucleic acid extraction or purification kit Zhejiang Hanwei Technology Co., Ltd. RevertAid First Strand cDNA Synthesis Kit Thermo Fisher Scientific TaqMan Fast Advanced Master Mix Thermo Fisher Scientific Hanwei RNA Extraction Kit Zhejiang Hanwei Technology Co., Ltd. RNALater Thermo Fisher Scientific Human Lipoprotein A ELISA Kit Abcam Shutai 50 VIK FRANCE LIMITED

[0387] Table 8 Main instruments and equipment

[0388]

[0389]

[0390] Example 1 Construction of LPA transgenic cell line:

[0391] (1.1) Construction of LPA expression plasmid

[0392] The human LPA gene mRNA sequence (NM_005577.4) was obtained from NCBI, and Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform full gene synthesis, and the gene was inserted into the multiple cloning site of the pcDNA3.1 vector to construct the pcDNA3.1-hLPA expression vector. The plasmid was amplified, extracted, and endotoxin removed for the subsequent construction of a stable cell line.

[0393] (1.2) Cell transfection

[0394] (1.2.1) Cell seeding

[0395] HEK-293A cells in logarithmic phase were digested and diluted to 2×10 5 cell / mL cell suspension, inoculated in 25cm 2 In a plastic culture flask, 5 mL of cell suspension was inoculated into each flask and cultured overnight. When the cell confluence rate was about 70%, transfection was performed.

[0396] (1.2.2) Transient cell transfection

[0397] When the cell confluence rate is about 70%, transfection is performed. Before transfection, remove the culture medium in the bottle, add 5mL OPTI-MEM culture medium for standby use, add 3μg of pcDNA3.1-hLPA plasmid DNA to 500μL OPTI-MEM culture medium, mix well; add 20μL Lipo2000 to another 500μL OPTI-MEM, mix gently, and let stand at room temperature for 5 minutes; mix the DNA suspension and Lipo2000 suspension to a total volume of 1mL, mix gently, and let stand at room temperature for 20 minutes; add the DNA and Lipo2000 mixture to a 25cm2 cell bottle, shake the culture bottle back and forth and left and right to mix, and place it in an incubator for culture. After transfection for 4 hours, replace the culture medium with 10ml complete culture medium.

[0398] (1.3) Screening of stable cell lines

[0399] (1.3.1) Cell screening

[0400] 48 hours after transfection, the cells were screened with a medium containing G418 (optimal screening concentration). The screening medium was replaced every 2-3 days. When normal cells died completely, the cells were cultured with a new medium without G418. The cell growth status was observed every day. When the cells reached 60% confluence, the cells were screened again with a medium containing G418 (optimal screening concentration).

[0401] (1.3.2) Cell culture and cryopreservation

[0402] When the cells reach a confluence rate of more than 90%, transfer the cells to a culture flask for continued culture, and then screen them every 4-5 days with a culture medium containing G418 (optimal screening concentration). Samples can be collected until a certain number of cells stably expressing the transfected plasmid are reached (about 15 days after transfection). After the screening is completed, when continuing to culture, the concentration of G418 added to the culture medium is reduced by half, and the cells are expanded and cultured for subsequent screening experiments or frozen.

[0403] Example 2 Evaluation of in vitro activity of lipoprotein (a) (Lp(a)) siRNA

[0404] In this example, the HEK293-LPA stable cell line prepared in Example 1 was used to evaluate the inhibitory activity of LPA target siRNAs RX001001 to RX001145 on the target gene LPA, with RX000001 used as a negative control.

[0405] Preparation of test samples: After centrifugation of each siRNA test sample, add appropriate amount of DEPC H2O according to the specifications of each tube to dissolve it and prepare it into a 20μM stock solution. Then, the stock solution is further diluted with DEPC H2O to 0.04μM and 0.004μM working solutions. The dose screening test is carried out with a final duplex concentration of 1nM and 0.1nM.

[0406] 96-well transfection and detection: Use trypsin to digest the HEK293-LPA stable cell line grown to near confluence, wash the cells, prepare the cell suspension, and use it for 96-well plate plating. Disperse 5μL of 0.04μM and 0.004μM working solutions in 20μL Opti-MEM to form an siRNA mixture, and disperse 0.2μL RNAiMAX in 24.8μL Opti-MEM. After incubation for 5 minutes, mix with each siRNA mixture and incubate for 10 minutes to form a transfection complex. Add 150μL of cell suspension (12,000 cells) to the transfection complex in each well, and place the 96-well plate in the incubator for further culture for 24h.

[0407] The 96-well plate was removed and the cells were lysed. Total RNA was extracted using a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction. A reverse transcription kit (ThermoFisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) 18 reverse transcription primer was selected. According to the method described in the reverse transcription kit manual, 20 μL reverse transcription system was configured and the reverse transcription reaction was completed. Then, the expression of the target gene mRNA in the cell was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Roche, LightCycler480). In the real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is used as an internal reference gene, and primers for the target gene and the GAPDH internal reference gene are used to detect the target gene and the GAPDH internal reference gene, respectively. The sequences of the detection primers are shown in Table 9.

[0408] Table 9 Sequences of detection primers

[0409]

[0410] In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group.

[0411] Table 10 Inhibitory activity of target genes in HEK293-LPA stable cell lines after administration of RX000001 to RX001145

[0412]

[0413]

[0414]

[0415] Example 3 Evaluation of the activity of L96 vector-conjugated siRNA in HDI mice

[0416] In this example, the BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same LPA target siRNA sense strand 3' end conjugated with L96 vector conjugates RZ001002 to RZ001030 on the target gene LPA, with RZ000002 used as a negative control.

[0417] Plasmid construction:

[0418] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to construct the LPA gene sequence (NM_005577.4) into the pcDNA-CMV vector plasmid to form the pcDNA-CMV-RG001 expression plasmid.

[0419] Mouse model construction:

[0420] The BALB / c mouse hydrodynamic injection model is a model established by rapidly injecting the pcDNA-CMV-RG001 plasmid solution into the mouse through the tail vein of the mouse. On the third day of the experiment, the mouse was injected with 10μg pcDNA-CMV-RG001 through the tail vein hydrodynamics within 5 seconds, and the injection volume was 8% of the mouse body weight. The plasmid DNA for injection was diluted with physiological saline, and the solution was prepared before injection and stored at 4.

[0421] Animal grouping, drug administration and tissue sample collection:

[0422] 6-8 week old BALB / c mice were randomly divided into groups according to body weight (all female), with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution of 0.1 mg / mL (calculated as siRNA), and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3), and 5 mice in all groups were killed on the 4th day (recorded as D4). The mice were grossly dissected and the liver tissue of each mouse was collected, and the liver tissue was cut into small pieces of about 2 mm3 and preserved with RNA later.

[0423] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then the total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0424] For each mouse, 1 μg of total RNA was taken respectively, and a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) 18 reverse transcription primer was selected. According to the method recorded in the reverse transcription kit manual, 20 μL reverse transcription system was configured and the reverse transcription reaction was completed. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then, the expression of the target gene mRNA in the animal was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In the real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as the internal reference gene, and the primer for the target gene and the primer for the Nero internal reference gene were used to detect the target gene and the Nero internal reference gene respectively. The sequence of the detection primer is shown in Table 11.

[0425] Table 11 Sequences of detection primers

[0426]

[0427] According to the method described in the instruction manual of the real-time fluorescence quantitative PCR kit, 10 μL of Real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the above reverse transcription reaction, 5 μL of TaqMan TMFast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 11), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system is placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and a two-step method is used for Real-time PCR amplification. The amplification program is 50 2min, then 95 pre-denaturation for 20s, 95 denaturation for 3s, 60 annealing and extension for 30s, and the process of denaturation, annealing and extension is repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group are relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0428] Table 12 Inhibitory activity of target genes in mice after administration of siRNA conjugates

[0429]

[0430]

[0431] The results of Example 3 show that at a dose of 1 mg / kg, RZ001003, RZ001004, RZ003005 and RZ001019 have an LPA mRNA inhibitory effect of greater than 50%.

[0432] Example 4 Evaluation of multiple dose activity of L96 vector conjugate siRNA in HDI mice

[0433] In this example, the BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the LPA target siRNA sense chain 3' end conjugated to the L96 vector conjugate RZ001002, RZ001003, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001019, RZ001026, and RZ001028 on the target gene LPA at different doses.

[0434] Animal grouping, drug administration and tissue sample collection:

[0435] BALB / c mice aged 6-8 weeks were randomly divided into groups (all female) according to body weight, with 5 mice in each group. The mouse HDI model was established by the same pcDNA-CMV-RG001 plasmid and mouse hydrodynamic injection model construction method as in Example 3. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were given a single dose of drug conjugates in the form of PBS solutions of 0.05mg / mL, 0.1mg / mL, and 0.3mg / mL (calculated as siRNA) by subcutaneous injection in the abdomen, and the administration volume was 10mL / kg of mouse body weight, that is, the dosage of each drug conjugate was 0.5mg / kg, 1mg / kg, and 3mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3), and 5 mice of all groups were killed on the 4th day (recorded as D4). The mice were grossly dissected and the liver tissues of each mouse were collected. The liver tissues were cut into small pieces of about 2 mm3 and preserved in RNA later.

[0436] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then the total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0437] For each mouse, 1 μg of total RNA was taken and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo(dT) was selected. 18Reverse transcription primers, according to the method described in the reverse transcription kit manual, configure 20 μL reverse transcription system and complete the reverse transcription reaction. After the reaction is completed, 60 μL RNase-Free water is added to the reverse transcription system to obtain a cDNA solution. Then the expression of the target gene mRNA in the animal is detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone is used as an internal reference gene, and the primer for the target gene and the primer for the Nero internal reference gene are used to detect the target gene and the Nero internal reference gene respectively. The sequence of the detection primer is shown in Table 10.

[0438] According to the method described in the instruction manual of the real-time fluorescence quantitative PCR kit, 10 μL of Real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the above reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 11), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system is placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and a two-step method is used for Real-time PCR amplification. The amplification program is 50 2min, then 95 pre-denaturation for 20s, 95 denaturation for 3s, 60 annealing and extension for 30s, and the process of denaturation, annealing and extension is repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group are relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0439] The results of Example 3 show that RZ001002, RZ001003, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001019, RZ001026, and RZ001028 can dose-dependently inhibit the expression of LPA mRNA in mouse liver tissue; at a dose of 3 mg / kg, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001026, and RZ001028 reduced the LPA mRNA expression by 80% ( Figure 1 , Table 13).

[0440] Table 13 Inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates

[0441]

[0442]

[0443] Example 5 Toxicological evaluation of L96 vector-conjugated siRNA in mice

[0444] In this example, a single subcutaneous injection of 300 mg / kg was used to evaluate the nature and extent of the toxicity of the carrier conjugates RZ001003, RZ001004, RZ001005, RZ001018, and RZ001026 in ICR mice.

[0445] Animal grouping, drug administration and tissue sample collection:

[0446] 6-8 week old ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 10 mice in each group, half male and half female. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 30 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 300 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate) and observed for 14 days. The day of administration was recorded as day 0 (recorded as D0), and clinical observations were performed at least once a day after the first administration during the test period, and body weight and food intake were measured twice a week; blood was taken from all animals on the 14th day of the test (recorded as D14) (mice were fasted for at least 12 hours before sampling and testing, and water was not forbidden), and hematology and blood biochemistry tests were performed. Blood was collected and anticoagulated with EDTA-K2, and routine blood tests were performed using an automatic modular blood and body fluid analyzer (Sysmex XN-10B1); blood samples (without anticoagulation) were collected and placed at room temperature for about 30 minutes. After the blood coagulated, it was centrifuged at 2000g for 10 minutes at 4°C, and blood biochemistry tests were performed using a fully automatic biochemical analyzer (Sysmex BX-4000). All animals were dissected on the 14th day of the experiment (denoted as D14). The mice to be dissected were fasted but not watered for at least 12 hours before dissection and anesthetized with Zotal 50 (Vicki Co., Ltd., France, 8G4VA). After blood collection, the animals were bled from the abdominal aorta to be euthanized, and gross anatomical observations were performed; histopathological examinations were performed on the livers and kidneys of all animals (using hematoxylin-eosin staining). The results are as follows Figure 2 As shown, the test substances had no significant effects on the general condition, body weight, and gross anatomical observation of food intake of ICR mice. In the RZ001005 300 mg / kg group, glandular duct-like tissue formation, fibrous tissue hyperplasia, and inflammatory cell infiltration were observed; in the RZ001026 300 mg / kg group, slight perivascular hepatocyte necrosis and inflammatory cell infiltration were observed; slight increase in mitosis was observed in the livers of some animals in each group; no pathological changes related to the test substances were observed in the kidneys.

[0447] Example 6 Evaluation of the activity of CR01008 vector-conjugated siRNA in HDI mice

[0448] In this example, the BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of siRNA conjugates such as RZ001031, the 3' end of the siRNA sense chain of the same LPA target site conjugated to the CR01008 vector conjugate, on the target gene LPA, with RZ000001 as a negative control and Olpasiran as a positive control (the sense chain of Olpasiran is (NAG25)sCmsAmGmCmCmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGms(invdA), with a nucleotide sequence as shown in SEQ ID NO.303; the antisense chain is UmsCfsGmUfAmUfAmAmCmAmAmUfAmAfGmGfGmGfCmsUfsGm, with a nucleotide sequence as shown in SEQ ID NO.304).

[0449] Animal grouping, drug administration and tissue sample collection:

[0450] BALB / c mice aged 6-8 weeks were randomly divided into groups (all female) according to body weight, with 5 mice in each group. The mouse HDI model was established by the same pcDNA-CMV-RG001 plasmid and mouse hydrodynamic injection model construction method as in Example 3. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose of all mice was calculated according to body weight, and a single administration was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution of 0.1 mg / mL (calculated as siRNA), and the administration volume was 10 mL / kg of mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3), and 5 mice of all groups were killed on the 4th day (recorded as D4). The mice were grossly dissected and the liver tissues of each mouse were collected. The liver tissues were cut into small pieces of about 2 mm3 and preserved in RNA later.

[0451] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then the total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0452] For each mouse, 1 μg of total RNA was taken and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo(dT) was selected. 18 Reverse transcription primers, according to the method described in the reverse transcription kit manual, configure 20 μL reverse transcription system and complete the reverse transcription reaction. After the reaction is completed, 60 μL RNase-Free water is added to the reverse transcription system to obtain a cDNA solution. Then the expression of the target gene mRNA in the animal is detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone is used as an internal reference gene, and the primer for the target gene and the primer for the Nero internal reference gene are used to detect the target gene and the Nero internal reference gene respectively. The sequence of the detection primer is shown in Table 10.

[0453] According to the method described in the instruction manual of the real-time fluorescence quantitative PCR kit, 10 μL of Real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the above reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 10), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system is placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and a two-step method is used for Real-time PCR amplification. The amplification program is 50 2min, then 95 pre-denaturation 20s, 95 denaturation 3s, 60 annealing and extension 30s, and the process of denaturation, annealing and extension is repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group are relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0454] Table 14 Inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates

[0455]

[0456]

[0457] The results of Example 6 showed that at a dose of 1 mg / kg, different modified CR01008 vector conjugate siRNAs could significantly inhibit LPA mRNA expression in animal liver tissue, among which RZ001031, RZ001032, RZ001033, and RZ001034 were better than Olpasiran in reducing mRNA levels. Figure 3 , Table 14).

[0458] Example 7 Construction of hApo(a) transgenic mice

[0459] hApo(a) transgenic mice were developed by Saiye (Suzhou) Biotechnology Co., Ltd. based on its unique Platform-based construction of conditional knock-in human Apo(a) transgenic mice.

[0460] (1) Design and construction of targeting vector: The human LPA gene was constructed into the vector to form a CAG promoter-loxP-PGK-Neo-6*SV40 pA-loxP-Kozak-Human LPA CDS-rBG pA targeting vector, which can insert the human LPA gene (NM_005577.4) into the intergenic region between the Eif4enif1 and Drg1 genes at the Hipp11 locus on mouse chromosome 11 in vivo. The targeting vector was verified by restriction digestion, PCR and sequencing, and plasmid extraction and linearization were performed after the targeting vector was verified.

[0461] (2) Electroporation of the targeting vector into embryonic stem cells and screening of positive clones: The targeting vector was electroporated into mouse embryonic stem cells (ES), and Neo was used for positive selection and DTA was used for negative selection. Drug-resistant clones were selected, and ES cells were verified by PCR, karyotype analysis, and Southern blot.

[0462] (3) Mouse preparation: Positive ES cells are microinjected and the injected blastocysts are transplanted into pseudo-pregnant female mice. The mice are identified after birth to obtain TurboKnockout mice with reproductive transmission ability.

[0463] (4) Obtaining hApo(a) transgenic mice: Mice expressing Cre specifically in the liver were hybridized with TurboKnockout mice, the resistance gene in the middle of the loxP site was removed, and the expression of the human LPA gene was activated to obtain hApo(a) transgenic mice.

[0464] Example 8 Evaluation of the protein-lowering effect of CR01008 vector-conjugated siRNA in hApo(a) transgenic mice

[0465] In this example, enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of Apo(a) protein in the serum of mice at different time points after a single administration of CR01008 vector conjugates RZ001031, RZ001032, RZ001033, RZ001034, RZ001035, RZ001038, RZ001039 and RZ0030044, with Olpasiran as a control.

[0466] Animal grouping, drug administration and tissue sample collection:

[0467] Apo(a) transgenic mice (Suzhou Xuanjing Biotechnology Co., Ltd. commissioned Saiye (Suzhou) Biotechnology Co., Ltd. to construct) were grouped according to the level of Apo(a) in serum. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PB11), and the sera of mice in all groups were collected on the 28th day (denoted as D28), the 35th day (denoted as D35), the 42nd day (denoted as D42), the 49th day (denoted as D49), the 56th day (denoted as D56), the 63rd day (denoted as D63), and the 70th day (denoted as D70), and the Apo(a) protein expression was determined using the human Lipoprotein A ELISA kit (Abcam, ab212165).

[0468] Table 15 Changes in human Apo(a) protein levels in transgenic mice after administration of siRNA

[0469]

[0470]

[0471] The results of Example 8 show that at a single dose of 1 mg / kg, RZ001031, RZ001032, RZ001033, RZ001034, RZ001035, RZ001038, and RZ001039 can all significantly reduce the Apo(a) protein level in Apo(a) transgenic mice; it was observed that on the 28th day, RZ001032 reduced the Apo(a) protein level by 90%, while Olpasiran reduced the Apo(a) protein effect by 75%; it was observed that on the 56th day, RZ001032 reduced the Apo(a) protein level by 50%, while Olpasiran reduced the Apo(a) protein effect by 30%; it was observed that on the 70th day, the Apo(a) protein level in the RZ001032 group had not yet returned to the pre-drug value. ( Figure 4 , Table 15).

[0472] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A double-stranded oligonucleotide for inhibiting LPA gene expression, characterized in that: The double-stranded oligonucleotide comprises a sense strand and an antisense strand; Wherein, the positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO.1, wherein the substantially identical means that the nucleotide sequence has no more than 3 nucleotide differences from the at least 15 consecutive nucleotides; Wherein, the antisense strand comprises at least 17 nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantially complementary strand means that the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides in the duplex region; Each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides; Optionally, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotides, wherein n is selected from 1 or 2, and R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; If R1 contains a substituent, the substituent in R1 refers to a C1-C6 alkoxy group or one or more hydrogen atoms on a C1-C6 alkoxy group are replaced by a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides; Optionally, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The antisense strand is complementary or substantially complementary to 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the nucleotide sequence of SEQ ID NO.1, and the substantially complementary refers to that the mismatch in the complementary region does not exceed 3 nucleotides.

3. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises 15, 16, 17, 18, 19 or 20 consecutive nucleotides in the sequence of SEQ ID NO.1 or a nucleotide sequence having no more than 2 nucleotide differences from the consecutive nucleotides; Optionally, the duplex region is 15-25 nucleotide pairs in length.

4. A double-stranded oligonucleotide for inhibiting LPA gene expression, characterized in that: The double-stranded oligonucleotide comprises a sense strand and an antisense strand; In the direction from the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17 or at least 18 consecutive nucleotides in any one of SEQ ID NOs. 147 to 291, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2 or 3 nucleotides; The sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; the substantially complementary sequence means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

5. The double-stranded oligonucleotide according to claim 4, characterized in that The nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of any one of SEQ ID NOs. 147 to 291, or a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides; Optionally, the antisense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 147 to 291; Optionally, the antisense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NOs. 147 to 291; Optionally, the antisense strand comprises a nucleotide sequence having 0 or 1 nucleotide difference from any one of SEQ ID NOs. 147 to 291; Optionally, the antisense strand is selected from or comprises any one of the nucleotide sequences in SEQ ID NOs. 147-291.

6. The double-stranded oligonucleotide according to claim 4, characterized in that The nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides of any one of SEQ ID NOs. 2 to 146, or differs from the consecutive nucleotides by 1, 2 or 3 nucleotides; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 2 to 146; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NOs. 2 to 146; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0 or 1 nucleotide difference from any one of SEQ ID NOs. 2 to 146; Optionally, the nucleotide sequence of the sense strand is selected from or comprises any one of SEQ ID NOs. 2 to 146.

7. The double-stranded oligonucleotide according to claim 4, characterized in that The double-stranded oligonucleotide is selected from the modified or unmodified double-stranded oligonucleotide shown in any number from RN001001 to RN001145 in Table 1; Optionally, the double-stranded oligonucleotide is selected from one or more of the modified or unmodified double-stranded oligonucleotides shown in the following numbers: RN001008, RN001010, RN001011, RN001025, RN001042, RN001074 and RN001121.

8. The double-stranded oligonucleotide according to claim 4, characterized in that Each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides; Optionally, substantially all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides; Optionally, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides; Optionally, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-optionally substituted C1-C6 alkyl modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of PNA, MNA, BNA, LNA, GNA, TNA, and UNA; If the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide contains a substituent, the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide means that one or more hydrogen atoms on the C1-C6 alkyl group are replaced by a substituent, and the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Wherein, n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; if R1 contains a substituent, the substituent in R1 means that one or more hydrogen atoms on the C1-C6 alkyl or C1-C6 alkoxy are replaced by a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each R2 is independently selected from an optionally substituted C1-C6 alkyl group; if R2 contains a substituent, the substituent in R2 means that one or more hydrogen atoms on the C1-C6 alkyl group are replaced by a substituent, and the substituent in R2 is independently selected from a C1-C3 alkyl group, a C1-C3 alkoxy group or a halogen; optionally, the 2'-halogenated modified nucleotide is selected from a 2'-fluoro modified nucleotide; Optionally, the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is selected from 2'-O-methyl modified nucleotides; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotide, 2'-O-ethoxymethyl modified nucleotide or 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides; Optionally, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide; Optionally, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides or 2'-O-methoxyethyl modified nucleotides.

9. The double-stranded oligonucleotide according to claim 8, characterized in that The sense strand and / or the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotides or 2'-O-Si(R2)3 modified nucleotides; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotides.

10. The double-stranded oligonucleotide according to claim 4, characterized in that The sense strand or the antisense strand comprises a 3' overhang having at least 1 nucleotide; Optionally, the antisense strand comprises a 3' overhang of at least 1 nucleotide; Optionally, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the sense strand is 18-21 nt in length, and the antisense strand is 19-23 nt in length; Optionally, the sense strand is 19 nt in length and the antisense strand is 21 nt in length; Optionally, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end; Optionally, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

11. The double-stranded oligonucleotide according to any one of claims 4 to 10, characterized in that The structure of the duplex region is shown in formula (101): SS: 5'-(N)a'-(X)i'-(N)b'-(X)t'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)i-(N)b-(X)t-(N)c-5' formula (101) Among them, SS represents the sense strand and AS represents the antisense strand; Each N is independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-deoxy modified nucleotide; Each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotide or 2'-O-Si(R2)3 modified nucleotide; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; each R2 is independently selected from optionally substituted C1-C6 alkyl; a, a', i, i', b, b', t, t', c, c', r', d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, i' is selected from an integer of 0-3, b' is selected from an integer of 4-13, t' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, i is selected from an integer of 0-1, b is selected from an integer of 4-8, t is selected from an integer of 0-4, and c is selected from an integer of 6-10; and i', t', r', i, t are not 0 at the same time, and 0≤t'+r'≤4; Optionally, a' is selected from an integer of 3-8, i' is selected from 0 or 1, b' is selected from an integer of 4-13, t' is selected from 0 or 1, c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, i is selected from 1, b is selected from an integer of 4-8, t is selected from 0 or 1, and c is selected from an integer of 6-10; Optionally, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides; Optionally, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide; Optionally, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide; Optionally, the sense strand is 18-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, at most two nucleotides of the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9 to 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(10): (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 9 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; (9) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the direction from 5' end to 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds; and / or, the 5' end and 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

12. The double-stranded oligonucleotide according to claim 11, characterized in that Each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and the sense strand is selected from any one of the following A1 to A11; and / or, the antisense strand is selected from any one of the following B1 to B41: The positive strand: from the 5' end to the 3' end, A1: GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm; A2: GmsCmsAmGmCmUmCfCfUfUfAmUmUmGmUmUmAmUmAm; A3: GmsCmsUmCmCmUmUfAfUfUfGmUmUmAmUmAmCmGmAm; A4: CmsGmsGmUmAmAmUfGfGfAfCmAmGmAmGmUmUmAmUm; A5: CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm; A6: AmsGmsAmGmGmAmCfAfAfCmAmGmAmAmUmAmUmUmAm; A7: AmsGmsAmGmG(moe)AmCfAfAfCmAmGmAmAmUmAmUmUmAm; A8: AmsGmsAmGmGmAmCfAfAfCfAmGmAmAmUmAmUmUmAm; A9: CmsUmsGmAmCmAmCfAfAfUfGmCmUmCmAmGmAmAmAm; A10: CmsUmsGmAmC(moe)AmCfAfAfUfGmCmUmCmAmGmAmAmAm; A11: CmsUmsGmAmCmAmCfAfAfUfGmC(moe)UmCmAmGmAmA(moe)Am; The antisense strand: In the direction from the 5'-end to the 3'-end, B1: AmsGfsUmAmGmUfUmCmAmUmGfAmUmCfAmAfGmCmCmsAmsGm; B2: AmsGfsUmAmGmUfUmCmAmUmGfAmUmCfA(moe)AfGmCmCmsAmsGm; B3: UmsAfsUmAmAmCfAmAmUmAmAfGmGmAfGmCfUmGmCmsCmsAm; B4: UmsAfsUmAmAmCfAmAmUmAmAfGmGmAfG(moe)CfUmGmCmsCmsAm; B5: UmsCfsGmUmAmUfAmAmCmAmAfUmAmAfGmGfAmGmCmsUmsGm; B6: AmsUfsAmAmCmUfCmUmGmUmCfCmAmUfUmAfCmCmGmsUmsGm; B7: AmsUfsAmAmCmUfCmUmGmUmCfCmAmUfT(moe)AfCmCmGmsUmsGm; B8: UmsAfsGmUmUmCfUmUmGmAmUfCmAmAfGmCfCmAmGmsCmsAm; B9: UmsAfsGmUmUmCfUmUmGmAmUfCmAmAfG(moe)CfCmAmGmsCmsAm; B10: UmsAfsAmUmAmUfUmCmUmGmUfUmGmUfCmCfUmCmUmsGmsAm; B11: UmsAfsAmUmAmUfUmCmUmGmUfUmGmUfC(moe)CfUmCmUmsGmsAm; B12: UmsUfsUmCmUmGfAmGmCmAmUfUmGmUfGmUfCmAmGmsAmsUm; B13: UmsUfsUmCmUmGfAmGmCmAmUfUmGmUfG(moe)UfCmAmGmsAmsUm; B14: AmsGfsUmAmGmUfUmCmAfUmGmAmUmCfAmAfGmCmCmsAmsGm; B15: AmsGfsUmAmGmUfUmCmAmUmGmAfUmCfAmAfGmCmCmsAmsGm; B16: AmsGfsUmAmGmUfUmCmAfUmGmAmUmCfA(moe)AfGmCmCmsAmsGm; B17:AmsGfsUmAmGmUfUmCmAmUmGmAfUmCfA(moe)AfGmCmCmsAmsGm; B18:UmsAfsUmAmAmCfAmAmUfAmAmGmGmAfGmCfUmGmCmsCmsAm; B19:UmsAfsUmAmAmCfAmAmUmAmAmGfGmAfGmCfUmGmCmsCmsAm; B20:UmsAfsUmAmAmCfAmAmUfAmAmGmGmAfG(moe)CfUmGmCmsCmsAm; B21:UmsAfsUmAmAmCfAmAmUmAmAmGfGmAfG(moe)CfUmGmCmsCmsAm; B22:UmsCfsGmUmAmUfAmAmCfAmAmUmAmAfGmGfAmGmCmsUmsGm; B23:UmsCfsGmUmAmUfAmAmCmAmAmUfAmAfGmGfAmGmCmsUmsGm; B24:UmsCfsGmUmAmUfAmAmCfAmAmUmAmAfG(moe)GfAmGmCmsUmsGm; B25:UmsCfsGmUmAmUfAmAmCmAmAmUfAmAfG(moe)GfAmGmCmsUmsGm; B26:AmsUfsAmAmCmUfCmUmGfUmCmCmAmUfUmAfCmCmGmsUmsGm; B27:AmsUfsAmAmCmUfCmUmGmUmCmCfAmUfUmAfCmCmGmsUmsGm; B28:AmsUfsAmAmCmUfCmUmGfUmCmCmAmUfU(moe)AfCmCmGmsUmsGm; B29:AmsUfsAmAmCmUfCmUmGmUmCmCfAmUfU(moe)AfCmCmGmsUmsGm; B30:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGmsCmsAm; B31:UmsAfsGmUmUmCfUmUmGmAmUmCfAmAfGmCfCmAmGmsCmsAm; B32:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfG(moe)CfCmAmGmsCmsAm; B33:UmsAfsGmUmUmCfUmUmGmAmUmCfAmAfG(moe)CfCmAmGmsCmsAm; B34:UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfCmCfUmCmUmsGmsAm; B35: UmsAfsAmUmAmUfUmCmUmGmUmUfGmUfCmCfUmCmUmsGmsAm; B36: UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfC(moe)CfUmCmUmsGmsAm; B37: UmsAfsAmUmAmUfUmCmUmGmUmUfGmUfC(moe)CfUmCmUmsGmsAm; B38: UmsUfsUmCmUmGfAmGmCfAmUmUmGmUfGmUfCmAmGmsAmsUm; B39: UmsUfsUmCmUmGfAmGmCmAmUmUfGmUfGmUfCmAmGmsAmsUm; B40: UmsUfsUmCmUmGfAmGmCfAmUmUmGmUfG(moe)UfCmAmGmsAmsUm; B41:UmsUfsUmCmUmGfAmGmCmAmUmUfGmUfG(moe)UfCmAmGmsAmsUm; Wherein, C, G, U, A, T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, thymidine-3'-phosphate, respectively; m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine modified nucleotide; the combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl modified nucleotide; s represents that the two nucleotides are connected by a thiophosphate backbone; Optionally, the double-stranded oligonucleotide is selected from any one of the following combinations of the sense strand and the antisense strand: RX001149 to RX001192: RX001149: A1 and B1; RX001150: A1 and B2; RX001151: A2 and B3; RX001152: A2 and B4; RX001153: A3 and B5; RX001154: A4 and B6; RX001155: A4 and B7; RX001156: A5 and B8; RX001157: A5 and B9; RX001158: A6 and B10; RX001159: A6 and B11; RX001160: A7 and B11; RX001161: A9 and B12; RX001162: A9 and B13; RX001163: A10 and B13; RX001164: A11 and B13; RX001165: A1 and B14; RX001166: A1 and B15; RX001167: A1 and B16; RX001168: A1 and B17; RX001169: A2 and B18; RX001170: A2 and B19; RX001171: A2 and B20; RX001172: A2 and B21; RX001173: A3 and B22; RX001174: A3 and B23; RX001175: A3 and B24; RX001176: A3 and B25; RX001177: A4 and B26; RX001178: A4 and B27; RX001179: A4 and B28; RX001180: A4 and B29; RX001181: A5 and B30; RX001182: A5 and B31; RX001183: A5 and B32; RX001184: A5 and B33; RX001185: A8 and B34; RX001186: A8 and B35; RX001187: A8 and B36; RX001188: A8 and B37; RX001189: A9 and B38; RX001190: A9 and B39; RX001191: A9 and B40; RX001192: A9 and B41.

13. The double-stranded oligonucleotide according to claim 4, characterized in that The dual oligonucleotide is selected from siRNA.

14. A conjugate, characterized in that The conjugate comprises the double-stranded oligonucleotide of any one of claims 1 to 13 and one or more ligands capable of binding to a cell surface receptor; Optionally, the 3' end and / or 5' end of the sense strand and / or the antisense strand is conjugated to the ligand; Optionally, the 3' end and / or the 5' end of the sense strand is conjugated to the ligand; Optionally, the 3' end of the sense strand is conjugated to the ligand; Optionally, the ligand is linked to the 3' end and / or 5' end of the sense strand and / or antisense strand via a phosphate group or a phosphorothioate group; Optionally, the number of the ligands is selected from 1, 2, 3 or 4; In an optional embodiment, the number of the ligands is selected from 1; In an optional embodiment, the number of the ligands is selected from 1, and one of the ligands is conjugated to the 3' end of the sense strand; Optionally, the ligand specifically targets liver tissue; Optionally, the ligand is selected from asialoglycoprotein receptor ligands; Optionally, the ligand comprises galactose or a derivative thereof; Optionally, the derivative of galactose is selected from N-acetylgalactosamine.

15. The conjugate according to claim 14, characterized in that The structure of the conjugate is shown in formula (201): Wherein, Nu represents the double oligonucleotide; Each R ligand independently selected from ASGPR ligands; y is selected from 1, 2, 3 or 4; each of said R ligand are independently conjugated to the 3' end and / or 5' end of the sense strand and / or the antisense strand, and each of the R ligand The conjugation positions are different; Optionally, y is selected from 1, 2, 3 or 4; Optionally, y is selected from 1 or 2; Optionally, y is selected from 1; Optionally, y is selected from 1, one of said R ligand conjugated to the 3' end of the sense strand; Optionally, y is selected from 2; Optionally, y is selected from 2, two of said R ligand conjugated to the 3' and 5' ends of the sense strand, respectively.

16. The conjugate according to claim 15, characterized in that Each R ligand Each is independently selected from the structure represented by formula (301) or a pharmaceutically acceptable salt or a stereoisomer thereof: Wherein, * represents the conjugation site of the ligand on the sense strand or the antisense strand; j is selected from 1, 2, 3 or 4; Each Z is independently selected from hydroxyl or thiol; Each p is independently selected from 1, 2 or 3; Each q is independently selected from 1, 2 or 3; Each R3 is independently selected from H, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group; if R3 contains a substituent, the substituent in R3 means that one or more hydrogen atoms on the C1-C6 alkyl group or the C1-C6 alkoxy group are replaced by a substituent, and the substituent in R3 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each L is independently selected from optionally substituted C1-C 20 Alkylene or R La and R La are independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5; if L contains a substituent, the substituent in L refers to C1-C 20 Alkylene or C 1-10 One or more hydrogen atoms on the alkylene group are substituted by a substituent, and the substituent in L is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each Y is independently selected from O, S or NH; Optionally, j is selected from 1, 2 or 3; Optionally, j is selected from 3; Optionally, Z is selected from hydroxyl; Optionally, p is selected from 1; Optionally, q is selected from 1; Optionally, each R3 is selected from H; Optionally, each L is independently selected from C1-C 10 Alkylene or Among them, R La and R La Independently selected from C1-C5 alkylene, k is 1, 2 or 3; Optionally, k is selected from 1; Optionally, each L is independently selected from Optionally, Y is selected from O; Optionally, each R ligand Independently selected from the structure shown in formula (302) or a pharmaceutically acceptable salt or a stereoisomer thereof: Optionally, each R ligand Independently selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof: Optionally, each Rligand is independently selected from any of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof:

17. The conjugate according to claim 15, characterized in that The Nu is selected from any one of RX001149 to RX001192; Optionally, Nu is selected from any one of RX001149 to RX001192, the ligand is selected from the formula (CR01008×3), y=1, and one of the ligands is conjugated to the 3′ end of the sense strand of the double-stranded oligonucleotide.

18. Use of the double-stranded oligonucleotide according to any one of claims 1 to 13 or the conjugate according to any one of claims 14 to 17 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene; Optionally, the disease or condition mediated by the LPA gene comprises a cardiovascular disease.

19. A pharmaceutical composition, characterized in that Comprising the double-stranded oligonucleotide according to any one of claims 1 to 13 and / or the conjugate according to any one of claims 14 to 17.

20. A kit, characterized in that The kit comprises the double-stranded oligonucleotide according to any one of claims 1 to 13, and / or the conjugate according to any one of claims 14 to 17, and / or the pharmaceutical composition according to claim 19.

21. A method for inhibiting LPA gene expression, characterized in that: The method comprises administering to a subject the double-stranded oligonucleotide of any one of claims 1 to 13, and / or the conjugate of any one of claims 14 to 17, and / or the pharmaceutical composition of claim 19; Optionally, the method comprises contacting a subject's cells with the double-stranded oligonucleotide, and / or the conjugate, and / or the pharmaceutical composition.

22. A method for alleviating, treating and / or preventing a disease or condition mediated by LPA, characterized in that: It comprises administering to a subject the double-stranded oligonucleotide of any one of claims 1 to 13, and / or the conjugate of any one of claims 14 to 17, and / or the pharmaceutical composition of claim 19; Optionally, the LPA-mediated disease or condition comprises a disease associated with mRNA levels expressed from an LPA gene; Optionally, the LPA-mediated disease or condition is a cardiovascular disease; Optionally, the cardiovascular disease includes: hyperlipoprotein(a)emia, Buerger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or pathology associated with elevated levels of Lp(a) containing particles.

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